2.4. Reference Spectra and Spectral Library

Reference spectra were identified from single-label samples for each fluorescent species: Cal 520, NucBlue, and cellular autofluorescence (unlabeled cells). Spectral images were acquired for each single-label sample, and images were corrected to a flat spectral response, as described above. Regions of interest were then selected corresponding to areas of intense, but not oversaturated, signals using the freehand selection region tool in ImageJ software [50]. The pixel-averaged spectrum from each region was extracted using the Image → Stacks → Plot Z-axis Profile command in ImageJ. This served as the reference spectrum for each label. Reference spectra were compiled in Excel (Microsoft Corporation, Redmond, WA, USA) and subsequently transferred to MATLAB (The MathWorks, Inc., Natick, MA, USA) as a spectral library for use with the analysis algorithms described below.